The present invention relates to novel phosphoramidites, including positive and neutrally charged compounds. The present invention also provides charge tags for attachment to materials including solid supports and nucleic acids, wherein the charge tags increase or decrease the net charge of the material. The present invention further provides methods for separating and characterizing molecules based on the charge differentials between modified and unmodified materials.
Methods for the detection and characterization of specific nucleic acid sequences and sequence variations have been used to detect the presence of viral or bacterial nucleic acid sequences indicative of an infection and to detect the presence of variants or alleles of genes associated with disease and cancers. These methods also find application in the identification of sources of nucleic acids, as for forensic analysis or for paternity determinations. Various methods are known to the art that may be used to detect and characterize specific nucleic acid sequences and sequence variants. Nonetheless, with the completion of the nucleic acid sequencing of the human genome, as well as the genomes of numerous other organisms such as pathogenic organisms, the demand for fast, reliable, cost-effective and user-friendly tests for the detection of specific nucleic acid sequences continues to grow. Importantly, these tests must be able to create a detectable signal from samples that contain very few copies of the sequence of interest.
There are a number of techniques that have been developed for characterizing specific nucleic acid sequences. Examples of detection techniques include the xe2x80x9cTaqManxe2x80x9d or nick-translation PCR assay described in U.S. Pat. No. 5,210,015 to Gelfand et al. (the disclosure of which is herein incorporated by reference), the assays described in U.S. Pat. Nos. 4,775,619 and 5,118,605 to Urdea (the disclosures of which are herein incorporated by reference), the catalytic hybridization amplification assay described in U.S. Pat. No. 5,403,711 to Walder and Walder (the disclosure of which is herein incorporated by reference), the cycling probe assay described in U.S. Pat. Nos. 4,876,187 and 5,011,769 to Duck et al., the target-catalyzed oligonucleotide modification assay described in U.S. Pat. Nos. 6,110,677 and 6,121,001 to Western et al. (the disclosures of which are herein incorporated by reference), the SNP detection methods of Orchid Bioscience in U.S. Pat. No. 5,952,174 (the disclosure of which is herein incorporated by reference), the methods of U.S. Pat. No. 5,882,867 to Ullman et al. (the disclosure of which is herein incorporated by reference) the polymerase chain reaction (PCR) described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188 to Mullis and Mullis et al. (the disclosures of which are herein incorporated by reference) and the ligase chain reaction (LCR) described in U.S. Pat. Nos. 5,427,930 and 5,494,810 to Birkenmeyer et al. and Barany et al. (the disclosures of which are herein incorporated by reference). The above examples are intended to be illustrative of nucleic acid-based detection assays and do not provide an exhaustive list. Each of these techniques requires a detection step for detecting a reaction product that is indicative of a desired target nucleic acid (e.g., detection of cleavage products, extension products, etc.). While a number of advances have been made in the assay methods and detection instrumentation to improve the sensitively, speed, and cost of detection methods the art is still in need of further improved methods, compositions, and systems to make the assays more sensitive and efficient.
The present invention relates to novel phosphoramidites, including positive and neutrally charged compounds. The present invention also provides charge tags for attachment to materials including solid supports and nucleic acids, wherein the charge tags increase or decrease the net charge of the material. The present invention further provides methods for separating and characterizing molecules based on the charge differentials between modified and unmodified materials.
For example, the present invention provides a composition comprising a charge tag attached to a nucleic acid molecule (e.g., to a terminal end of a nucleic acid molecule). In some embodiments, the charge tag comprises a phosphate group and a positively charged moiety. In some preferred embodiments, the charge tag further comprises a dye. The present invention is not limited by the position of the individual modular components of the charge tag. For example, in some embodiments, the dye is positioned between the nucleic acid and the positively charged moiety, while in other embodiments, the positively charged moiety is positioned between the nucleic acid and the dye. The present invention is also not limited by the number of each type of component in the charge tag (e.g., the number of dyes, positively charged moieties, etc.). For example, in some embodiments, the charge tag comprises first and second positively charged moieties.
In some embodiments of the present invention, the charge tag has a net positive charge. For example, in some embodiments, the charge tag has a net positive charge of 1, 2, 3, etc. In some embodiments, the charge tag possesses a positive charge only under certain reaction conditions (e.g., pH 6-10).
In some embodiments, the charge tag further comprises one or more nucleotides. In some embodiments, the nucleic acid molecule to which the charge tag is attached contains a sequence that is complementary to a target nucleic acid. In some such embodiments, the one or more nucleotides in the charge are not complementary to the target nucleic acid. In other such embodiments, the nucleic acid comprises a first portion complementary to a target nucleic acid and a second portion that is not complementary to said target nucleic acid, wherein the charge tag is attached to the second portion of the nucleic acid (e.g., to a terminal end of the nucleic acid that is located in the second portion).
In some embodiments of the present invention, the nucleic acid and the charge tag have a combined net neutral charge, wherein the charge tag, in isolation, has a net positive charge. In other embodiments, the nucleic acid and the charge tag have a combined net negative charge, wherein the charge tag has a net positive charge.
The present invention is not limited by the nature of the positively charged moiety of the charge tag. Positively charged moieties include, but are not limited to primary amines, secondary amines, tertiary amines, ammonium groups, positively charged metal groups (e.g., caged ions attached to the charge tag through a linking group), and the like.
In some embodiments, the charge tag further comprises a positively charged phosphoramidite or a neutral phosphoramidite. The present invention is not limited by the nature of the positively charged phosphoramidite or the neutral phosphoramidite. For example, in some embodiments, the charge tags comprise a novel phosphoramidite of the present invention.
For example, the present invention provides a composition comprising a positively charged phosphoramidite. In some embodiments, the positively charged phosphoramidite contains one or more positively charged moieties including, but not limited to, primary amine groups, secondary amine groups, tertiary amine groups, ammonium groups, charged metal ions, and the like. In some embodiments, the phosphoramidite has a net positive charge of one. In some particularly preferred embodiments, the phosphoramidite has the structure: 
wherein, X is a reactive phosphate group (e.g., PO4) and Y is a protecting group (e.g., dimethoxy trityl [DMT]) and/or a protected group (e.g., DMT-protected hydroxyl group).
The present invention further provides a composition comprising a nucleic acid molecule containing a positively or neutrally charged phosphoramidite. The present invention also provides a composition comprising a charge tag attached to a terminal end of a nucleic acid molecule, wherein the charge tag comprises a positively charged or neutrally charged phosphoramidite. In some preferred embodiments, the positively charged phosphoramite comprises an amine group, wherein the amine group is not further attached to another molecule (a molecule other than the phosphoramidite).
The present invention further provides a composition comprising a neutrally charged phosphoramidite. In some preferred embodiments, the neutrally charged phosphoramidite comprises a nitrogen-containing chemical group selected from the group comprising primary amine, secondary amine, tertiary amine, ammonium group, and charged metal ion. In some embodiments, the composition further comprises a nucleic acid molecule attached to the neutrally charged phosphoramidite. In some preferred embodiments, the nucleic acid molecule is attached to a charge tag comprising the neutrally charged phosphoramidite. The charge tag may further comprise, in any order, other components. For example, the charge tag may further comprise a positively charged phosphoramidite. In some embodiments of the present invention, the charge tag containing the neutrally charged phosphoramidite has a net positive charge. In some particularly preferred embodiments of the present invention, the neutrally charged phosphoramidite has the structure: 
wherein X is a protecting group (e.g., dimethoxy trityl group [DMT]) and/or a protected group (e.g., DMT-protected hydroxyl group), Z is a reactive phosphate, and N comprises an amine group. In some preferred embodiments, the N group is Nxe2x80x94(CH2)nCH3, wherein n is 0 or a positive integer from 1 to 12.
The present invention also provides a composition comprising a solid support attached to a charge tag. For example, in some embodiments, the charge tag comprises a positively charged moiety and a reactive group configured to allow the charge tag to covalently attach to a nucleic acid molecule. Any of the charge tags described herein, may be attached to the solid support.
The present invention further provides a composition comprising a fluorescent dye directly bonded to a phosphate group, wherein the phosphate group is directly bonded to an amine group. In some embodiments, the composition comprises a charge tag, wherein the fluorescent dye is contained within the charge tag. The present invention is not limited by the nature of the fluorescent dye. However, in some preferred embodiments, the fluorescent dye comprises a Cy dye (e.g., Cy3).
The present invention also provides a mixture comprising a plurality of oligonucleotides attached to charge tags. In some embodiments, each oligonucleotide is attached to a different charge tag. In other embodiments, two or more different oligonucleotides have the same type of charge tag. In some preferred embodiments, each of the charge tags comprises a phosphate group and a positively charged moiety. While not limited by the number of oligonucleotides attached to different charge tags, in some embodiments, the plurality of oligonucleotides comprises four or more oligonucleotides (e.g., 5, 6, 7, . . . , 10, . . . , 50, . . . , 100, . . . ), each attached to a different charge tag. Any of the charge tags described herein are contemplated for use in the mixtures.
The present invention further provides a method of separating nucleic acid molecules, comprising the steps of: a) treating a charge-balanced oligonucleotide containing a charge tag under conditions such that a charge-unbalanced oligonucleotide containing the charge tag is produced, wherein the charge-unbalanced oligonucleotide is contained in a reaction mixture; and b) separating the charge-unbalanced oligonucleotide from the reaction mixture. While the present invention is not limited by the means by which a charge-unbalanced oligonucleotide is generated, in some preferred embodiments, the oligonucleotides are treated with a reactant (e.g., a nuclease). Any of the charge tags described herein are contemplated for use in the method. While the present invention is not limited by the nature of the separation step, contemplated separation steps include, but are not limited to, gel electrophoretic separation, capillary electrophoretic separation, capillary zone electrophoretic separation, and separation is a microchannel.
The present invention also provides a method of separating nucleic acid molecules, comprising the steps of: a) treating a plurality of charge-balanced oligonucleotides, each containing different charge tags, under conditions such that two or more charge-unbalanced oligonucleotides containing the charge tags are produced, wherein the charge-unbalanced oligonucleotides are contained in a reaction mixture; and b) separating the charge-unbalanced oligonucleotides from the reaction mixture. In some preferred embodiments, the separating comprises separating the charge-unbalanced oligonucleotides such that charge-unbalanced oligonucleotides containing different charge tags are separated from one another. Any of the charge tag, oligonucleotide mixtures, and separation methods described herein may be used with this method.
Definition
To facilitate an understanding of the present invention, a number of terms and phrases are defined below:
The term xe2x80x9ccharge-balancedxe2x80x9d molecule or oligonucleotide refers to a molecule or oligonucleotide (the input oligonucleotide in a reaction) that has been modified such that the modified molecule or oligonucleotide bears a charge, such that when the modified molecule or oligonucleotide is either reduced in size (e.g., cleaved, shortened, disassociated, unbound, or otherwise altered such that it is part of a complex or molecule having a lower aggregate molecular weight) or increased in sized (e.g., enlarged, elongated, associated, bound, or otherwise altered such that it is part of a complex or molecule having a higher aggregate molecular weight), a resulting product bears a net charge or charge to mass ratio different from the input molecule or oligonucleotide (the resulting molecule thus being a xe2x80x9ccharge-unbalancedxe2x80x9d molecule or oligonucleotide) thereby permitting separation of the input and reacted molecules or oligonucleotides on the basis of charge. The term xe2x80x9ccharge-balancedxe2x80x9d does not imply that the modified or balanced molecule or oligonucleotide has a net neutral charge (although this can be the case). Charge-balancing refers to the design and modification of a molecule or oligonucleotide such that a specific reaction product generated from this input molecule or oligonucleotide can be separated on the basis of charge from the input molecule or oligonucleotide.
For example, in an INVADER oligonucleotide-directed cleavage assay in which the probe oligonucleotide bears the sequence: 5xe2x80x2 TTCTTTTCACCAGCGAGACGGG 3xe2x80x2 (ie., SEQ ID NO:1 without the modified bases) and cleavage of the probe occurs between the second and third residues, one possible charge-balanced version of this oligonucleotide would be: 5xe2x80x2 Cy3-AminoT-Amino-TCTTTTCACCAGCGAGAC GGG 3xe2x80x2 (SEQ ID NO:1). This modified oligonucleotide bears a net negative charge. After cleavage, the following oligonucleotides are generated: 5xe2x80x2 Cy3-AminoT-Amino-T 3xe2x80x2 and 5xe2x80x2 CTTTCACCAGCGAGACGGG 3xe2x80x2 (residues 3-22 of SEQ ID NO:l). 5xe2x80x2 Cy3-AminoT-Amino-T 3xe2x80x2 bears a detectable moiety (the positively charged Cy3 dye) and two amino-modified bases. The amino-modified bases and the Cy3 dye contribute positive charges in excess of the negative charges contributed by the phosphate groups and thus the 5xe2x80x2 Cy3-AminoT-Amino-T 3xe2x80x2 oligonucleotide has a net positive charge. The other, longer cleavage fragment, like the input probe, bears a net negative charge. Because the 5xe2x80x2 Cy3-AminoT-Amino-T 3xe2x80x2 fragment is separable on the basis of charge from the input probe (the charge-balanced oligonucleotide), it is referred to as a charge-unbalanced oligonucleotide. The longer cleavage products are not generally separated on the basis of charge from the input oligonucleotide as both oligonucleotides bear a net negative charge.
The term xe2x80x9cnet neutral chargexe2x80x9d when used in reference to a molecule or oligonucleotide, including modified oligonucleotides, indicates that the sum of the charges present (e.g., R-NH3+ groups on thymidines, the N3 nitrogen of cytosine, presence or absence or phosphate groups, etc.) under the desired reaction or separation conditions is essentially zero. A molecule or oligonucleotide having a net neutral charge would not migrate in an electrical field.
The term xe2x80x9cnet positive chargexe2x80x9d when used in reference to a molecule or oligonucleotide, including modified oligonucleotides, indicates that the sum of the charges present (e.g., R-NH3+ groups on thymidines, the N3 nitrogen of cytosine, presence or absence or phosphate groups, etc.) under the desired reaction conditions is +1 or greater. A molecule or oligonucleotide having a net positive charge would migrate toward the negative electrode in an electrical field.
The term xe2x80x9cnet negative chargexe2x80x9d when used in reference to a molecule or oligonucleotide, including modified oligonucleotides, indicates that the sum of the charges present (e.g., R-NH3+ groups on thymidines, the N3 nitrogen of cytosine, presence or absence or phosphate groups, etc.) under the desired reaction conditions is xe2x88x921 or lower. A molecule or oligonucleotide having a net negative charge would migrate toward the positive electrode in an electrical field.
As used herein, the terms xe2x80x9ccomplementaryxe2x80x9d or xe2x80x9ccomplementarityxe2x80x9d are used in reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) related by the base-pairing rules. For example, for the sequence xe2x80x9c5xe2x80x2-A-G-T-3xe2x80x2,xe2x80x9d is complementary to the sequence xe2x80x9c3xe2x80x2-T-C-A-5xe2x80x2.xe2x80x9d Complementarity may be xe2x80x9cpartial,xe2x80x9d in which only some of the nucleic acids"" bases are matched according to the base pairing rules. Or, there may be xe2x80x9ccompletexe2x80x9d or xe2x80x9ctotalxe2x80x9d complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid strand, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand.
The term xe2x80x9chomologyxe2x80x9d and xe2x80x9chomologousxe2x80x9d refers to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence.
As used herein, the term xe2x80x9chybridizationxe2x80x9d is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the Tm of the formed hybrid. xe2x80x9cHybridizationxe2x80x9d methods involve the annealing of one nucleic acid to another, complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the xe2x80x9chybridizationxe2x80x9d process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modem biology.
With regard to complementarity, it is important for some diagnostic applications to determine whether the hybridization represents complete or partial complementarity. For example, where it is desired to detect simply the presence or absence of pathogen DNA (such as from a virus, bacterium, fungi, mycoplasma, protozoan) it is only important that the hybridization method ensures hybridization when the relevant sequence is present; conditions can be selected where both partially complementary probes and completely complementary probes will hybridize. Other diagnostic applications, however, may require that the hybridization method distinguish between partial and complete complementarity. It may be of interest to detect genetic polymorphisms. For example, human hemoglobin is composed, in part, of four polypeptide chains. Two of these chains are identical chains of 141 amino acids (alpha chains) and two of these chains are identical chains of 146 amino acids (beta chains). The gene encoding the beta chain is known to exhibit polymorphism. The normal allele encodes a beta chain having glutamic acid at the sixth position. The mutant allele encodes a beta chain having valine at the sixth position. This difference in amino acids has a profound (most profound when the individual is homozygous for the mutant allele) physiological impact known clinically as sickle cell anemia It is well known that the genetic basis of the amino acid change involves a single base difference between the normal allele DNA sequence and the mutant allele DNA sequence.
The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5xe2x80x2 end of one sequence is paired with the 3xe2x80x2 end of the other, is in xe2x80x9cantiparallel association.xe2x80x9d Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
As used herein, the term xe2x80x9cTmxe2x80x9d is used in reference to the xe2x80x9cmelting temperature.xe2x80x9d The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. Several equations for calculating the Tm of nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(%G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other references (e.g., Allawi, H. T. and SantaLucia, J., Jr. Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36, 10581-94 (1997) include more sophisticated computations which take structural and environmental, as well as sequence characteristics into account for the calculation of Tm.
As used herein the term xe2x80x9cstringencyxe2x80x9d is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With xe2x80x9chigh stringencyxe2x80x9d conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of xe2x80x9cweakxe2x80x9d or xe2x80x9clowxe2x80x9d stringency are often required when it is desired that nucleic acids which are not completely complementary to one another be hybridized or annealed together.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides and more preferably at least about 15 to 30 nucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof.
Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5xe2x80x2 phosphate of one mononucleotide pentose ring is attached to the 3xe2x80x2 oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the xe2x80x9c5xe2x80x2 endxe2x80x9d if its 5xe2x80x2 phosphate is not linked to the 3xe2x80x2 oxygen of a mononucleotide pentose ring and as the xe2x80x9c3xe2x80x2 endxe2x80x9d if its 3xe2x80x2 oxygen is not linked to a 5xe2x80x2 phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5xe2x80x2 and 3xe2x80x2 ends. A first region along a nucleic acid strand is said to be upstream of another region if the 3xe2x80x2 end of the first region is before the 5xe2x80x2 end of the second region when moving along a strand of nucleic acid in a 5xe2x80x2 to 3xe2x80x2 direction.
The term xe2x80x9clabelxe2x80x9d as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal, and that can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable.
The term xe2x80x9csamplexe2x80x9d in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin.
Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc.
Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
The term xe2x80x9csource of target nucleic acidxe2x80x9d refers to any sample that contains nucleic acids (RNA or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to cultures, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, semen, and animal or plant tissue.
As used herein, the term xe2x80x9ccharge tagxe2x80x9d refers to a modular chemical complex that is attached to or to be attached to another molecule, wherein the charge tag has a net charge that differs from the net charge of the other molecule. For example, charge tags may be attached to nucleic acid molecules (e.g., to the terminal end of a nucleic acid molecule). Charge tags contain any number of desired components including, but not limited to, dyes, linker groups, nucleotides, phosphoramidites, phosphonates, phosphate groups, amine groups, fluorescent quencher groups and the like.
In a xe2x80x9cmixture comprising a plurality of oligonucleotides with each oligonucleotide attached to a different charge tag,xe2x80x9d two or more oligonucleotides each possess a distinct charge tag, wherein the chemical makeup of the charge tags differ from one another. A mixture of oligonucleotides, each with a different charge tag, may also comprise additional oligonucleotides. For example, the mixture may contain a first set of oligonucleotides, each with identical first charge tags and a second set of oligonucleotides, each with an identical second charge tags.
As used herein, the term xe2x80x9cpositively charged moietyxe2x80x9d refers to a chemical group or molecule that contains a net positive charge. Positively charged moieties may be attached to or associated with other molecules or materials. A composition containing a positively charged moiety may itself have a net positive charge (because of the positively charged moiety or otherwise), but need not. In some embodiments of the present invention, positively charged moieties include, but are not limited to, amines (e.g., primary, secondary, and tertiary amines). For example, in some embodiments of the present invention, phosphoramidites contain a positively charged moiety comprising an amine. Amine groups are often used as linking chemistries for attaching to or more molecules (e.g., attaching a phosphoramidite to another molecule). However, in some embodiments of the present invention, amine groups are not used as linking groups, but are provided to give a molecule a positive charge. Thus, in some embodiments, the amines are attached to a molecule of interest (e.g., a phosphoramidite), but are not further attached to another molecule (e.g., are not attached to a molecule other than the phosphoramidite).
As used herein, the term xe2x80x9cdyexe2x80x9d refers to a molecule, compound, or substance that can provide an optically detectable signal (e.g., fluorescent, luminescent, colorimetric, etc). For example, dyes include fluorescent molecules that can be associated with nucleic acid molecules (e.g., Cy3).
As used herein, the term xe2x80x9cprotecting groupxe2x80x9d refers to a molecule or chemical group that is covalently attached to a compound to prevent chemical modification of the compound or modification of specific chemical groups of the compound. For example, protecting groups may be attached to a reactive group of a compound to prevent the reactive group from participating in chemical reactions including, for example, intramolecular reactions. In some cases, a protecting group may act as a leaving group, such that when the molecule is added to another compound in a desired synthesis reaction, the protecting group is lost, allowing a reactive group to participate in covalent bonding to the compound. The phosphoramidites of the present invention typically contain one or more protective groups prior to their addition to nucleic acid molecules. For example, the reactive phosphate of the phosphoramidite (i.e., the phosphate group that is covalently attached to another molecule when the phosphoramidite is added to the other molecule) may contain one or more protecting groups. A detailed description of phosphoramidites and their addition to nucleic acid molecules is provided Beaucage and Iyer (Tetrahedron 49:1925 [1993]), herein incorporated by reference in its entirety.
As used herein, the terms xe2x80x9csolid supportxe2x80x9d or xe2x80x9csupportxe2x80x9d refer to any material that provides a solid or semi-solid structure with which another material can be attached. Such materials include smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) as well as textured and porous materials. Such materials also include, but are not limited to, gels, rubbers, polymers, and other non-rigid materials. Solid supports need not be flat. Supports include any type of shape including spherical shapes (e.g., beads). Materials attached to solid support may be attached to any portion of the solid support (e.g., may be attached to an interior portion of a porous solid support material). Preferred embodiments of the present invention have biological molecules such as nucleic acid molecules, charge tags, and proteins attached to solid supports. A biological material is xe2x80x9cattachedxe2x80x9d to a solid support when it is associated with the solid support through a non-random chemical or physical interaction. In some preferred embodiments, the attachment is through a covalent bond. However, attachments need not be covalent or permanent. In some embodiments, materials are attached to a solid support through a xe2x80x9cspacer moleculexe2x80x9d or xe2x80x9clinking group.xe2x80x9d Such spacer molecules are molecules that have a first portion that attaches to the biological material and a second portion that attaches to the solid support. Thus, when attached to the solid support, the spacer molecule separates the solid support and the biological materials, but is attached to both.
As used herein, the term xe2x80x9cdirectly bonded,xe2x80x9d in reference to two molecules refers to covalent bonding between the two molecules without any intervening linking group or spacer groups that are not part of parent molecules.
As used herein, the terms xe2x80x9clinking groupxe2x80x9d and xe2x80x9clinker groupxe2x80x9d refer to an atom or molecule that links or bonds two entities (e.g., solid supports, oligonucleotides, or other molecules), but that is not a part of either of the individual linked entities.
As used herein, the term xe2x80x9creactant,xe2x80x9d when referring to an agent that is used to generate charge-unbalanced molecules from charge-balanced molecules, refers to any agent (e.g., enzyme, chemical, physical device, etc.) that can alter a charge-balanced molecule such that a charge-unbalanced molecule is created.
As used herein, the methods of xe2x80x9ccapillary electrophoresis,xe2x80x9d xe2x80x9ccapillary zone electrophoresis,xe2x80x9d and xe2x80x9cmicrofluidsxe2x80x9d refer to methods for use in the separation methods of the present invention. The methods of capillary electrophoresis, capillary zone electrophoresis, and microfluids are described in texts and journals including, but not limited to, Baker (1995) Capillary Electrophoresis, Wiley-Interscience, New York, New York, Weinberger (2000) Capillary Electrophoresis, Second Edition, Academic Press, San Deigo, California, Atamna et al., J. Liq. Chromatogr., 13:2517 (1990), Nishi et al., Anal. Chem., 61:2434 (1989), Terabe et al., Anal. Chem., 56:111 (1984), Bousse et al., Annu. Rev. Biophys. Biomol. Struct., 29:155 (2000), and U.S. Pat. Nos. 5,916,426, 5,807,682, 5,703,222, 5,470,705, 5,777,096, and 5,514,543, each of which is herein incorporated by reference in its entirety.
As used herein, the term xe2x80x9ckitxe2x80x9d refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term xe2x80x9cfragmented kitxe2x80x9d refers to a delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term xe2x80x9cfragmented kitxe2x80x9d is intended to encompass kits containing Analyte specific reagents (ASR""s) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contain a subportion of the total kit components are included in the term xe2x80x9cfragmented kit.xe2x80x9d In contrast, a xe2x80x9ccombined kitxe2x80x9d refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term xe2x80x9ckitxe2x80x9d includes both fragmented and combined kits.